Journal: International Journal of Molecular Medicine
Article Title: Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury
Figure Lengend Snippet: Digoxin inhibits platelet-derived growth factor (PDGF)-BB-induced adhesion molecule expression and effects the expression of key proteins in the extracelluar matrix in vascular smooth muscle cells (VSMCs). VSMCs were pre-cultured in serum-free medium for 24 h. The serum-starved VSMCs were then stimulated with PDGF-BB for 48 h in the absence or presence of digoxin (100 nM). The protein levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by western blot analysis. (A) One representative image out of 4 independently performed experiments is shown. (B–D) The graphs represented the relative level of these proteins for 4 independent experiment. ( # P
Article Snippet: The membranes were blocked, and then incubated with various antibodies overnight, such as anti-CDK4 (Cat. no. SC23896, 1:500), anti-CDK6 (Cat. no. SC53638, 1:500), anti-p27Kip1 (Cat. no. SC1641, 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SM22a (Cat. no. A5228, 1:1,000), anti-calponin (Cat. no. C2687, 1:1,000), anti-smooth muscle (SM) α-actin (Cat. no. A2172, 1:1,000) (all from Sigma), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; Tyr183/185; Cat. no. 5135, 1:1,000), anti-JNK (Cat. no. 9258 1:1,000), anti-p38 mitogen-activated protein kinase (MAPK; Cat. no. 9228, 1:1,000), anti-phospho-p38 MAPK (Thr180/Tyr182; Cat. no. 4092, 1:1,000; p-MAPK), anti-extracellular signal-regulated kinase (ERK)1/2 (Cat. no. 4696, 1:1,000), anti-phospho-ERK1/2 (p-ERK, Tyr204; Cat. no. 4374, 1:1000), anti-phospho-Akt (p-Akt, S473; Cat. no. 12694, 1:1,000), anti-Akt (Cat. no. 2920, 1:1,000), anti-phospho-GSK-3β (p-GSK-3β, S9; Cat. no. 14630, 1:1,000), anti-GSK-3β (Cat. no. 9832, 1:1,000) (all from Cell Signaling Technology), anti-ILK (Cat. no. 61183, 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), anti-intercellular adhesion molecule-1 (ICAM-1; Cat. no. 4915, 1:500), anti-vascular cell adhesion molecule-1 (VCAM-1; Cat. no. 13662, 1:500) (both from Cell Signaling Technology), anti-matrix metalloproteinase (MMP)-2 (Cat. no. SC13594, 1:500), anti-MMP-9 (Cat. no. SC21733, 1:500) anti-tissue inhibitors of metalloproteinase (TIMP)-1 (Cat. no. SC21734, 1:500), anti-TIMP-2 (Cat. no. SC365671, 1:500) (all from Santa Cruz Biotechnology, Inc.), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat. no. 5174, 1:2,500) and anti-β-actin (Cat. no. 8457, 1:2,500) (both from Cell Signaling Technology), and then with the horseradish peroxidase-conjugated secondary antibody (Beijing TDY Biotech Co., Ltd.) (1:5,000) for 2 h. Specific protein expression levels were normalized to GAPDH or β-actin for total protein analyses or to total proteins for phosphorylated protein measurements.
Techniques: Derivative Assay, Expressing, Cell Culture, Western Blot